Variation in the bioaccumulation of a sediment-sorbed hydrophobic compound by benthic macroinvertebrates: patterns and mechanisms

Aquatic ecological risk assessment is primarily focused on aqueous exposure, but many hydrophobic contaminants bind to particulate material and accumulate in sediments. The risk posed by such contaminants is partially dependent on the importance of dietary exposure. Here, we describe the bioaccumulation of a highly hydrophobic compound (dioctadecyl-dimethyl ammonium chloride (DODMAC)) to four freshwater macroinvertebrates (i.e., Asellus aquaticus, Chironomus riparius, Gammarus pulex, Lumbriculus variegatus) and investigate the mechanistic basis for observed interspecific variation in bioaccumulation. Although more than 99.99% of DODMAC was sediment-bound, it was bioavailable to all four species via dietary exposure. Interspecific variation in bioaccumulation was apparent despite the lack of selective feeding and biotransformation potential and after normalization for body size and lipid content. Chironomus riparius had the highest lipid-normalized DODMAC concentration and L. variegatus had the lowest. Study species differed in factors affecting uptake (i.e., feeding rate) and absorption efficiency (i.e., gut passage time and gut surfactancy). Feeding rate did not explain interspecific variation in bioaccumulation, but bioaccumulation was enhanced by either high surfactancy and short gut passage time (e.g., G. pulex) or low surfactancy and long gut passage time (e.g., C. riparius). Risk assessment of hydrophobic contaminants should consider dietary exposure and the potential food chain effects of interspecific variation in bioaccumulation.     

A field experiment to determine the relative importance of prey and water as sources of As, Cd, Co, Cu, Pb, and Zn for the aquatic invertebrate Sialis velata

We measured the relative importance of water and food as sources of the trace elements As, Cd, Co, Cu, Pb, and Zn for larvae of the benthic insect Sialis velata. To achieve this end, we first exposed its prey, the midge Chironomus riparius, to trace elements in a contaminated lake located near a nonferrous metal smelter in northwestern Quebec. Concentrations of five of the six trace elements reached a steady state in prey within one week. When predators were exposed to these contaminated prey, their trace element concentrations increased markedly for all of the trace elements studied. In contrast, exposure of S. velata to contaminated lake water led to no significant increase in their concentrations of five of the six trace elements ([Pb] increased only slightly). Additional treatments in which the predator was exposed to these trace elements in both food and water or in a combination of food and water and sediment did not lead to further increases in its contaminant concentrations beyond those measured in the food-only treatment. We conclude that food (prey) is the almost exclusive source of As, Cd, Co, Cu, and Zn for S. velata and that it is the source of 94% of its Pb. Model curves for trace element accumulation over time by the predator fit our measured data well, and trace element concentrations in food alone are sufficient to model the exchange of these contaminants in this insect.     

Sorption and desorption behavior of tributyltin with natural sediments

Tributyltin (TBT) sorption to four natural sediment samples in artificial seawater was examined under a range of modified pH and salinity conditions. Three of the sediment samples were relatively pristine with regard to TBT contamination, but the fourth was a TBT-contaminated sediment from a commercial marina. Sorption of TBT was described well by linear sorption isotherms, with distribution coefficients ranging from 6.1 to 5210 L/kg depending on the pH and salinity. The sediment organic C content and particle size distribution were important determinants of sorption behavior. The presence of resident TBT in the contaminated marina sediment caused a substantial reduction in further TBT sorption. Desorption of TBT from the marina sediment was described by relatively large observed distribution coefficients ranging from 5100 to 9400 L/kg, suggesting that aging effects may reduce sorption reversibility. Increased artificial seawater salinity generally reduced TBT sorption at pH 4 and 6, but enhanced TBT sorption at pH 8. Regardless of salinity, maximum sorption of TBT was observed at pH 6, which is attributed to an optimal balance between the abundance of cationic TBT+ species and deprotonated surface ligands. Consideration of aqueous TBT speciation along with octanol-water partitioning behavior suggests that hydrophobic partitioning of TBTCl(0) to nonpolar organic matter was important for pH < 6, while partitioning of TBTOH(0) was important at higher pH.     

The internal distribution of nickel and thallium in two freshwater invertebrates and its relevance to trophic transfer

Although nickel and thallium are present at potentially harmful concentrations in some lakes, there is little information on their bioaccumulation and transfer up aquatic food webs. To measure the propensity of animals for accumulating and transferring these contaminants along food chains, we exposed two common types of invertebrates, an insect (Chironomus riparius) and a worm (Tubifex tubifex), to these metals spiked into sediment. We then measured the subcellular distribution of Ni and Tl in these invertebrates to estimate the likelihood that these metals will have toxic effects on these prey or be transferred to higher trophic levels. In both species, at least half of their Ni and TI was present in fractions that are purportedly detoxified (granules and metal-binding proteins). Furthermore, based on information in the literature concerning prey subcellular fractions that are likely to be trophically available (TAM), we estimate that much of the Ni and TI in these animals (43-84%) is available for transfer to a predator. To test this prediction, we fed these invertebrates to the alderfly Sialis velata, and measured the efficiency with which this predator assimilated Ni and Tl from each prey type. The majority of both trace metals (58-83%) was assimilated by the predator, which suggests that these contaminants would be easily transferred along aquatic food chains and that models describing Ni and Tl accumulation by aquatic animals should consider food as a source of these metals. The proportion of metal that could potentially be taken up by a consumer (% TAM) and the actual percentage assimilated by S. velata fell on or reasonably close to a 1:1 line for the 4 prey-metal combinations.     

TBT causes regime shift in shallow lakes

Tributyltin (TBT) is an organotin compound used since the early 1960s as a biocide in boat antifouling paints. Its use has been linked to a host of negative effects in marine ecosystems including malformations and imposex in Mollusca and acute toxicity in many other aquatic animals. Yet, the consequences of TBT use in freshwaters are largely unknown. Here, for the first time we reveal that TBT may have caused hitherto unsuspected damage to freshwater ecosystems. Through an analysis of dated sediment cores collected from a system of recreationally boated, shallow lakes, we show that first evidence of TBT is associated with a dramatic loss of submerged vegetation and associated diverse animal communities. Cause and effect are difficult to unravel in our study. However, we hypothesize that TBT, through reducing populations of grazing organisms in lakes already affected by eutrophication, promoted the replacement of macrophytes by phytoplankton, ultimately leading to a regime shift in the ecosystem. Our findings may have parallels in freshwater ecosystems all over the world.     

Sub-lethal effects of copper to benthic invertebrates explained by sediment properties and dietary exposure

The next generation of sediment quality guidelines (SQGs) requires better established causal links between the chronic exposure and effects of metals from both dissolved and dietary sources. The potential for dietary exposure from sediment metals to cause toxic effects to benthic invertebrates is strongly influenced by the metal-binding properties of the sediments. For relatively oxidized sediments, sublethal effects of copper to the epibenthic deposit-feeding amphipod, Melita plumulosa, and the benthic harpacticoid copepod, Nitocra spinipes, were investigated. Effects on reproduction were strongly influenced by the properties of the sediments and sediment-bound copper was found to be the major contribution to the toxicity. For sediments with the same total copper concentrations, effects were less for sediments with greater concentrations of fine particles (<63 μm sediment) or particulate organic carbon (OC). The OC-normalized copper concentration in the <63 μm sediment fraction provided a single effects threshold for all sediment types. For M. plumulosa and N. spinipes, the 10% effect concentrations (EC10s) were 5.2 and 4.8 mg <63 μm Cu g(-1) OC. These chronic EC10s indicate that a SQG of 3.5 mg <63 μm Cu g(-1) OC, that was previously proposed based on a species sensitivity distribution of acute no effects thresholds data for 12 benthic organisms, will be protective for these species. The study confirms the appropriateness of using SQGs that vary with sediment properties and that SQGs of this form provide adequate protection for metal exposure via both dissolved and dietary exposure pathways.     

New model calculations of pH-depending tributyltin adsorption onto montmorillonite surface and montmorillonite-rich sediment

Interactions of the pollutant tributyltin (TBT) with mineral surfaces affects its distribution and transport in aqueous systems. In the present work, model calculations are reported that quantify TBT adsorption onto pure-phase montmorillonite (sample SWy) under various pH and salinity conditions that are important from an environmental perspective. The pH level in the system is of substantial interest because it affects the speciation of TBT in solution as well as the surface properties of the solid phase, which are both important for adsorption reactions. The model is based on the generalized diffused layer model that includes >X- sites in order to account for the cation exchange effects of TBT attraction. The presence of >AIOH and >SiOH sites atthe mineral surface was not considered separately during calculation. Instead, nonselective sites (>SOH) versus selective sites (>SsOH) were distinguished with respect to the sorptive sites on montmorillonite. The latter are characterized by a high affinity of TBT bonding. Both sorptive sites exhibit the same protolysis constants but different TBT binding constants [logK(TBT/2) = -1.18 for (>SOH), logK(TBT/1) = 3.98 for (>SsOH)]. LogK(X/TBT) for the cation exchange reaction was determined as between 3.05 and 4.14. The results indicate that the inclusion of selective sites during calculations is essential for quantifying pH-dependent TBT adsorption successfully. The parameters determined for the TBT adsorption onto pure-phase montmorillonite were subsequently used to calculate pH-dependent TBT adsorption onto a natural montmorillonite-rich sediment.     

Use of life cycle assessments to evaluate the environmental footprint of contaminated sediment remediation

Ecological and human risks often drive the selection of remedial alternatives for contaminated sediments. Traditional human and ecological risk assessment (HERA) includes assessing risk for benthic organisms and aquatic fauna associated with exposure to contaminated sediments before and after remediation as well as risk for human exposure but does not consider the environmental footprint associated with implementing remedial alternatives. Assessment of environmental effects over the whole life cycle (i.e., Life Cycle Assessment, LCA) could complement HERA and help in selecting the most appropriate sediment management alternative. Even though LCA has been developed and applied in multiple environmental management cases, applications to contaminated sediments and marine ecosystems are in general less frequent. This paper implements LCA methodology for the case of the polychlorinated dibenzo-p-dioxins and -furans (PCDD/F)-contaminated Grenland fjord in Norway. LCA was applied to investigate the environmental footprint of different active and passive thin-layer capping alternatives as compared to natural recovery. The results showed that capping was preferable to natural recovery when analysis is limited to effects related to the site contamination. Incorporation of impacts related to the use of resources and energy during the implementation of a thin layer cap increase the environmental footprint by over 1 order of magnitude, making capping inferior to the natural recovery alternative. Use of biomass-derived activated carbon, where carbon dioxide is sequestered during the production process, reduces the overall environmental impact to that of natural recovery. The results from this study show that LCA may be a valuable tool for assessing the environmental footprint of sediment remediation projects and for sustainable sediment management.     

Mechanistic models to perform population risk assessment with the midge Chironomus riparius: application to heavy metals

Mechanistic models can substantially contribute to population risk assessment to assess effects on population and to increase the relevance of the toxicity parameters estimated at an individual level. We use four mechanistic models to change the scale from concentration to effects on individuals and from individuals to population with the midge Chironomus riparius: a kinetics model; an energy-based effects model, linking effects on the life cycle and compound body residues; a matrix approach to derive population growth rate; and an energy-based population model to derive carrying capacity. The whole "model battery" was applied to cadmium and copper. The data came from growth, survival, and reproduction tests. We also incorporated information about compounds physiological mode of action and kinetics. Thresholds at population level were derived through comparisons with our control database. We showed that our two population endpoints (carrying capacity and population growth rate) provide complementary information about toxicity risks, even if, in our study, population growth rate appeared to be slightly more sensitive than carrying capacity. We found population no effect concentration of, respectively, 0.42 and 9.3 mg/kg for cadmium and copper. We also showed that information about physiological mode of action was relevant, whereas a kinetics test was unnecessary.     

Biology-based modeling to analyze uranium toxicity data on Daphnia magna in a multigeneration study

Recent studies have investigated chronic toxicity of waterborne depleted uranium on the life cycle and physiology of Daphnia magna. In particular, a reduction in food assimilation was observed. Our aims here were to examine whether this reduction could fully account for observed effects on both growth and reproduction, for three successive generations, and to investigate through microscope analyses whether this reduction resulted from direct damage to the intestinal epithelium. We analyzed data obtained by exposing Daphnia magna to uranium over three successive generations. We used energy-based models, which are both able to fit simultaneously growth and reproduction and are biologically relevant. Two possible modes of action were compared - decrease in food assimilation rate and increase in maintenance costs. In our models, effects were related either to internal concentration or to exposure concentration. The model that fitted the data best represented a decrease in food assimilation related to exposure concentration. Furthermore, observations of consequent histological damage to the intestinal epithelium, together with uranium precipitates in the epithelial cells, supported the assumption that uranium has direct effects on the digestive tract. We were able to model the data in all generations and showed that sensitivity increased from one generation to the next, in particular through a significant increase of the intensity of effect, once the threshold for appearance of effects was exceeded.     

Optimisation of the formulation and of the technological process of egg-based products for the prevention of Salmonella enteritidis survival and growth

The aim of this work was to study the survival of Salmonella enteritidis after a pressure treatment in relation to compositive variables (NaCl content, pH), both in model and real systems, the latter consisting in an egg-based mayonnaise type product. Moreover, the fate of the surviving cells of S. enteritidis has been monitored during storage at 10 degrees C and the growth or death parameters have been calculated and modelled in relation to pH, NaCl concentration of the medium and entity of the pressure treatment applied. The modelling of the effects of the environmental factors on the treatment effectiveness indicated that the salt content and pH displayed a synergistic effect with pressure, whose extent was higher in the mayonnaise based products than in BHI. In fact, while in the model systems the cell recovery and growth during the subsequent incubation at 10 degrees C was allowed in many combinations of the Central Composite Design, in the real systems no recovery or growth of S. enteritidis were observed. This viability loss, which was maximum at pH 4.00 or 2% NaCl, could not be attributed merely to the interactions of such variables, but probably involves the naturally occurring antimicrobial enzymes of the raw material, whose activity can be enhanced by the pressure treatment.     

Ecotoxicological effects of activated carbon amendments on macroinvertebrates in nonpolluted and polluted sediments

Amendment of contaminated sediment with activated carbon (AC) is a remediation technique that has demonstrated its ability to reduce aqueous concentrations of hydrophobic organic compounds. The application of AC, however, requires information on possible ecological effects, especially effects on benthic species. Here, we provide data on the effects of AC addition on locomotion, ventilation, sediment avoidance, mortality, and growth of two benthic species, Gammarus pulex and Asellus aquaticus , in clean versus polycyclic aromatic hydrocarbon (PAH) contaminated sediment. Exposure to PAH was quantified using 76 μm polyoxymethylene passive samplers. In clean sediment, AC amendment caused no behavioral effects on both species after 3-5 days exposure, no effect on the survival of A. aquaticus , moderate effect on the survival of G. pulex (LC(50) = 3.1% AC), and no effects on growth. In contrast, no survivors were detected in PAH contaminated sediment without AC. Addition of 1% AC, however, resulted in a substantial reduction of water exposure concentration and increased survival of G. pulex and A. aquaticus by 30 and 100% in 8 days and 5 and 50% after 28 days exposure, respectively. We conclude that AC addition leads to substantial improvement of habitat quality in contaminated sediments and outweighs ecological side effects.     

Reproductive effects of long-term exposure to Bisphenol A in the fathead minnow (Pimephales promelas)

Bisphenol A (BPA), a high-volume chemical used to make polycarbonate plastic, epoxy resins, and other chemicals has been reported to be weakly estrogenic. To investigate the effects of long-term exposure to Bisphenol A, a multigeneration study was conducted in which fathead minnows (Pimephales promelas) were exposed to water concentrations of BPA in the range from 1 to 1280 micrograms/L. In this paper, we report the growth and reproductive effects of BPA on sexually mature adults in the F0 generation (after 43, 71, and 164 d of exposure) and the effects on hatchability in the F1 generation. Mean measured concentrations of BPA in the water for all doses, over a 164-d exposure period, were between 70% and 96% of nominal. An inhibitory effect of BPA on somatic growth (length and weight) occurred in adult male fish exposed to 640 and 1280 micrograms/L (after 71 and 164 d). BPA induced vitellogenin synthesis (VTG; a biomarker for estrogen exposure) in males at concentrations of 640 and 1280 micrograms/L after 43 d and 160 micrograms/L after 71 d. In females, plasma VTG concentrations were elevated above controls only after 164-d exposure to 640 micrograms/L. Inhibition of gonadal growth (as measured by the gonadosomatic index) occurred in both males and females at concentrations of 640 and 1280 micrograms/L after 164 d. In males, a concentration of 16 micrograms/L altered the proportion of sex cell types in the testis, suggesting inhibition of spermatogenesis. Concentrations of BPA that induced VTG synthesis and affected gonadal development were lower than those that resulted in discernible effects on reproductive output. Egg production was inhibited at a BPA concentration of 1280 micrograms/L, and hatchability in the F1 generation was reduced at a BPA concentration of 640 micrograms/L (there were not enough eggs spawned in the 1280 micrograms/L group for hatchability studies to be conducted). The results demonstrate that BPA acts as a weak estrogen to fish when administered via the water, with effects on breeding at and above 640 micrograms/L.     

Effect of high-pressure homogenization, nonfat milk solids, and milkfat on the technological performance of a functional strain for the production of probiotic fermented milks

The aim of this research was the evaluation of the effects of milkfat content, nonfat milk solids content, and high-pressure homogenization on 1) fermentation rates of the probiotic strain Lactobacillus paracasei BFE 5264 inoculated in milk; 2) viability loss of this strain during refrigerated storage; and 3) texture parameters, volatile compounds, and sensorial properties of the coagula obtained. The data achieved suggested a very strong effect of the independent variables on the measured attributes of fermented milks. In fact, the coagulation times were significantly affected by pressure and added milkfat, and the rheological parameters of the fermented milk increased with the pressure applied to the milk for added nonfat milk solids concentrations lower than 3%. Moreover, the polynomial models and the relative response surfaces obtained permitted us to identify the levels of the 3 independent variables that minimized the viability loss of the probiotic strain used during refrigerated storage.     

Comparison of five methods for measuring sediment toxicity of hydrophobic contaminants

Sediment toxicity from hydrophobic organic compounds (HOCs) is complicated by chemical partitioning among multiple phases and sediment-specific bioavailability. In this study, we used three hydrophobic pyrethroid insecticides as test compounds and derived 10-d median lethal concentrations (LC50s) for Chironomus tentans in three different sediments. The LC50s were expressed using HOC concentrations on a bulk sediment basis (C(S)), organic carbon (OC)-normalized sediment basis (C(S-OC)), porewater basis (C(PW)), dissolved organic carbon (DOC)-normalized porewater basis (C(PW-DOC)), and freely dissolved porewater basis (C(free)). The bulk phase C(S) and C(PW) yielded highly variable LC50s across sediment types, whereas the use of normalized concentrations C(S-OC) and C(PW-DOC) generally reduced variability due to sediment type but not that due to aging. In contrast, LC50s based on C(free) were essentially independent of sediment conditions. The sediment pore water samples contained approximately 20-90 mg L(-1) DOC, and the C(free) expressed as a percentage of the total bulk pore water concentration ranged from 9 to 28% for fenpropathrin (mean = 19%), 8 to 18% for bifenthrin (mean = 13%), and 3 to 8% for cyfluthrin (mean = 6%) across the different sediments. These results indicate thatthe use of C(free) reduces uncertainties caused by sediment variables such as OC properties and aging effects.     

Removal of copper from contaminated soil by use of poly(amidoamine) dendrimers

This study characterizes poly(amidoamine) (PAMAM) dendrimers of various generations and terminal functional groups for removal of copper(II) in a sandy soil. Effects of dendrimer dose, generation number, pH, terminal functional groups, and ionic strength on the removal efficiency were investigated through a series of column tests. Over 90% of copper initially sorbed in the soil was removed by use of approximately 66 bed volumes of 0.10% (w/w) of a generation 4.5 dendrimer with carboxylate terminal groups at pH 6.0. On the basis of equal equivalent dose, dendrimers of lower generation removed more copper. Lowering pH enhanced copper removal for all dendrimers tested. In contrast, types of terminal groups (carboxylate, amine, or hydroxyl) showed a modest effect on the removal efficiency. Results from a sequential extraction procedure suggested that dendrimers removed primarily exchangeable and carbonate-bound copper. The residual copper in treated soil is predominantly bound with soil organic matter (SOM), which is much less available physical-chemically or biologically. Spent dendrimers were recovered through nanofiltration with a commercially available nanofilter. Upon acid regeneration, recovered dendrimers were reused and performed as well as the virgin dendrimers. The dendrimers may be used as reusable, high-capacity extracting agents for in situ removal of heavy metals from contaminated soils.     

Multigeneration exposure of the springtail Folsomia candida to phenanthrone: from dose-response relationships to threshold concentrations

Results of life-cycle toxicity experiments are supposed to be indicative for long-term effects of exposure to toxicants. Several studies, however, have shown that adaptation or extinction of populations exposed for several generations may occur. The aim of this study was therefore to determine if the effects of the PAH phenanthrene on survival and reproduction of the springtail Folsomia candida exposed for 10 consecutive generations to contaminated soil would progressively increase, or, alternatively, if adaptation of the test organisms to the toxicant would occur. LC50 values for the first four generations were similar (171-215 micromol/kg dry soil), as expected for a narcotic compound. In the fourth generation, springtails exposed to a concentration similarto the EC50 for one generation (163 micromol/kg dry soil) showed internal phenanthrene concentrations in the range known to cause mortality; no reproduction took place, and the population went extinct. From the fifth generation onwards, survival and reproduction were not affected by the remaining exposure concentrations. Apparently, up to a certain threshold concentration (above 77 and below 163 micromol/kg dry soil), the springtails were able to metabolize phenanthrene, as shown by the lack of adverse effects and the lack of adaptation. During multigeneration exposure, the graded concentration-response relationship changed into an all-or-nothing response with a defined threshold concentration.Together with the worsening of effects, this raises concerns about the use of single-generation studies to tackle long-term population effects of environmental toxicants.     

Environmental dust exposure as a factor contributing to an increase in Escherichia coli O157 and Salmonella populations on cattle hides in feedyards

A study was conducted to determine the impact of exposure to dust in the cattle load-out area in feedyards on pathogen contamination of cattle hides. A total of 250 cattle hides were sampled during summer and fall months, which are associated with elevated prevalence of Escherichia coli O157 in West Texas. Animals were removed from their home pens and restrained in a chute and sampled prior to exposure to dust generated as a result of a simulated loading exercise. The cattle hides were sampled again after exposure to the loading dust to determine total numbers of pathogens on cattle hides on leaving their home pen (before loading) and on cattle hides after exposure to the dust in the loading area. Air and dirt samples from the home pens and the cattle load-out area were also collected. The presence of E. coli O157 and Salmonella was determined in all the samples, and when a positive sample was identified, the total numbers of these bacteria present were enumerated. The total numbers of pathogens increased after dust exposure; Salmonella counts increased from 1.09 log most probable number (MPN)/cm2 to 1.74 log MPN/cm2 after exposure, and E. coli O157 counts increased from 0.80 to 2.35 log MPN/cm2 after sampling. E. coli O157 and Salmonella were recovered from the air samples during dust generation at 6.66 and 11.1%, respectively. Salmonella and E. coli O157 prevalence was not changed and was not associated with the exposure to the dust. Results indicate airborne dust generated as a result of cattle movement and loading could be an important determining factor in total numbers of pathogens recovered on cattle hides.     

Prediction of the thickness loss and recovery of cut-pile carpets imposed to UV exposure and wear using polynomial regression method

Ultraviolet radiation which exists in environment around can be regarded as a major source of textile materials. In this study, the effect of UV exposure on the cut-pile carpet was investigated. A UV chamber was used to expose different levels of accelerated UV radiation on the samples of the machine-woven pile carpet. Wear test were then carried out on all samples using a Hexapod tumbler machine. The short-term static loading was applied to the carpets and the thickness loss percent (TL) and recovery percent (RP) of the samples at different times after load removal was measured using standard methods. Factorial experimental design and response surface method were applied for to create polynomial regression models and predict each of the thickness loss and recovery percent of carpet samples. The model is capable to determine the contribution of different variables. The results of the modeling revealed a desirable fit. The adjusted R² values were also high and significant. The ANOVA test indicated that the presented models were valid at 5% significant level.     

Modeling combined effects of pulsed exposure to carbaryl and chlorpyrifos on Gammarus pulex

Aquatic risk assessment can be improved if we are able to quantitatively predict the effects resulting from sequential pulsed exposure to multiple compounds. We evaluate two modeling approaches, both extended to suit multiple compounds, the semi-mechanistic threshold damage model (TDM), and a model based on time-weighted averages (TWA). The TDM predicts that recovery of damage to Gammarus pulex from exposure to chlorpyrifos takes longer than that from exposure to carbaryl and consequently that the sequence of exposure matters. We measured survival of the freshwater invertebrate Gammarus pulex after sequential pulsed exposure to carbaryl and chlorpyrifos. Two groups of organisms were exposed to a first pulse of either carbaryl or chlorpyrifos for 1 day and then, after a recovery period of two weeks, to a second pulse with the other compound. The comparison of mortalities caused by each pulse, as well as combined mortalities in both treatments, show that the sequence of exposure to pulses of contaminants does indeed matter. Previous exposure to chlorpyrifos leads to significantly increased mortality from subsequent pulses of carbaryl, but not the other way round. The TDM facilitates a process-based ecotoxicological explanation by simulating the recovery dynamics and outperforms the TWA model.